This invention relates to the use of tannin related compounds for the selective extraction and purification of mucilaginous polysaccharides from biological materials such as plants, ground biological tissues, or fermented cultured broths from microorganisms. In particular, this invention relates to the precipitation of acetylated mannose polymers derived from the aloe plant and beta glucans from oats and fungi.
Generally speaking, mucilaginous polysaccharides are defined as biopolymers characterized by hetero or polysaccharide chains, either linear or branched, having acetyl, nitrogen acetyl, or other nitrogen functional groups associated with the main polysaccharide chain, and containing protein chemically bound to one or more of the external OH groups of the main structure of the polysaccharide chains. Some of these mucilaginous polysaccharides are immunomodulators, and their biological and physical properties make them useful in a variety of applications as ingredients for cosmetics, beverages and pharmaceuticals and as viscosifiers in several multiple chemical production processes. Because of their complex native chemical structure, mucilaginous polysaccharides tend to form a colloidal network with other substances present in solution. It is difficult to separate or isolate these substances while at the same time retaining most or all of their native properties.
Aloe polysaccharides are known as acetylated hetero poly-mannose biopolymers having about one or more acetyl groups per saccharide. (Manna S., McAnalley B.H.; xe2x80x9cDetermination of the position of the )O-acetyl group in a beta- (1xe2x86x924)-mannan (acemannan) from Aloe barbadensis millerxe2x80x9d. Carbohydrate Research (1993) Mar 17; 241:317-319). Although the author takes for granted, without any previous carbohydrate analysis, that the sample that he was analyzing was 100% mannan in its composition, he concludes that the O-acetyl groups in Aloe polysaccharides are located at C-2/C-3 position and at C6 position in a 50:50 ratio.
The acetyl group : saccharide ratio in aloe polysaccharides can also vary with the age of the source plant and other environmental factors, but in general terms the plant in its natural state typically maintains the inner biopolymer with an acetyl group : per saccharide ratio of 1 or higher, wherein the polysaccharide is composed mainly, but not entirely, of mannose. It is believed that the biological and physical characteristics of aloe polysaccharides are attributable in large part to the presence of acetylated mannose residues. This biopolymer is different thanother poly-mannans such as locust bean gum or guar gum which have no reported immunological biological activity.
Mucilaginous polysaccharides have traditionally been isolated either by the use of organic solvents, the use of ammonium sulfate, quaternary ammonium salts and by the use of cationic detergents. However, some of these procedures tend to alter the initial chemical structure of the native biopolymer.
Polysaccharides and mucilaginous polysaccharides will generally form viscous solutions or dispersions exhibiting a typical non-newtonian viscosity profile in polar solvents due to hydrogen bonding (R. L. Whistler, xe2x80x9cIndustrial Gumsxe2x80x9dR. L. Whistler and J. N. B. Miller, eds. Academic Press Inc., New York, N.Y., 1959, p 1). Because of the general inability of polysaccharides to swell in organic liquids such as ethanol, methanol, or acetone, these organic solvents traditionally have been used to precipitate polysaccharides from their carrier solutions. However, aside from requiring large amounts of solvent, the solvent precipitation technique tends to provide, co-precipitation of other materials such as organic acids, certain salts, proteins, and other similar substances, giving low product yields and/a somewhat degraded biopolymer.
Ethanol is generally preferred for precipitating the mucilaginous polysaccharides network from aloe vera and other similar mucilages and polysaccharides. Typically, aqueous solutions or extracts of the mucilaginous polysaccharides are treated with five or more volumes of ethanol (U.S. Pat. Nos. 4,957,907, 4,917,890, 4,735,935) to precipitate the polysaccharide. This ethanol-based method of precipitating aloe polysaccharides tends to yield a final polysaccharide product having a significantly reduced acetyl group total saccharide ratio, which is different than the initial native chemical structure, and to denature the glycoproteins present in the hydroparenchima of aloe vera leaves. The reduced acetyl group: saccharide can be attributed to a variety of factors such as the time required for making the gel of aloe allowing enough time for hydrolytic enzymes present in the hydroparechima to act on the polysaccharide, and for the normal increase in temperature caused by the addition of ethanol to the aqueous extract. The second technique for isolating mucilaginous polysaccharides requires the use of large quantities of ammonium sulfate or quaternary ammonium salts to precipitate all the polysaccharides. Detergent cations, such as cetyltrimethylammonium (CTA) or cetylpyridium (CP) also have the ability to form insoluble salts with hydrophobic polyanions, and these insoluble salts then precipitate out from their aqueous solution (J. E. Scott, Chem and Industry (London) 1568 (1955), and A. S. Jones, Biochem. Biophys. Acta, 10, 607 (1953)). The use of CTA and other similar detergent cations for precipitating polysaccharides is another example of structural polysaccharide alteration. Dupont showed that different angiogenic biological activities were obtained from different samples of shark cartilage mucopolysacchardie recovered from initial water extracts, which were treated with different precipitation techniques. After in vivo and in vitro examination, only those shark cartilage mucopolysaccharides which were obtained using the technique of water extraction followed by molecular ultrafiltration, were able to show significant biological activity as compared with other shark cartilage polysaacharide samples obtained either by the classical solvent preciptation using ethanol or using detergent cations (Dupont,Eric et.al., U.S. Pat. No. 5,618,925: xe2x80x9cExtracts of shark cartilage having an anti-angiogenic activity and an effect on tumor regression; process of making thereof.xe2x80x9d Apr. 8,1997).
Another commonly used procedure for recovering polysaccharides is the use of ammonium salts. However this procedure works best when individual samples containing polysaccharides have similar ionic character. However, some biopolymers present in certain biological extracts often contain various varieties of polysaccharides, which can vary widely in ionic character. This variability makes the use of ammonium salts unsuitable for application in biological extracts containing heterogeneous types of biopolymers.
Tannins have been classified chemically either as (1) condensed tannins (known as proanthocyanidins), which are chemically defined as flavanoid-based polymers, or (2) hydrolyzable tannins (Haslam E., (1981). Vegetable tannins. In Conn, EE (ed.): xe2x80x9cThe biochemistry of plants Volume 7,xe2x80x9d New York Academic Press, p 527-556. In the case of condensed tannins, the beta ring of the flava monomer is generally substituted with two or three ortho-hydroxyl groups. An example of a condensed tannin is the one found in the testa of the grain Sorghum bicolor. On the other hand, hydrolyzable tannins are characterized by a polyhydroxy alcohol esterified with gallic acid (3,4,5-trihydroxybenzoic acid). Hydrolyzable tannins include the family of substances known as ellagitannins and gallotannins which, upon acid hydrolysis, give rise to ellagic and gallic acid. The typical commercial form of hydrolyzable tannins is known as tannic acid. It is well known that hydrolyzable tannins tend to form insoluble complexes with proteins. These complexes are generally water insoluble, but they can be dissociated by various techniques including solvation with organic solvents. Both condensed and hydrolyzable tannins can form insoluble complexes with biological protein-polysaccharide colloidal networks under certain conditions.
Four distinct mechanisms have been proposed to describe the chemistry of the interaction between proteins and tannins. These mechanisms are based on covalent interactions, ionic, and hydrogen bonding or hydrophobic interactions. Covalent interactions may result from nucleophilic attack of amino acid side chains such as lysine on the quinonoid oxidation products of tannin. (O-quinones formed in plant extracts, their reaction with amino acids and peptides. Pierpoint W E (1969), Biochem J. 112 : 609-618.) Such reactions occur most readily at high pH, where oxidation of the phenolic group is most likely. Ionic interactions between cationic amino acid side chains such as lysine and the phenolate anion occur only at pH values greater than the pKa of the phenolic hydroxyl group (pKa=9-11). Loomis W. D. (1974), xe2x80x9cOvercoming problems of phenolic and quinones in the isolation of plant enzymes and organellesxe2x80x9d. Meth Enz 31: 528-544. The most common mode of interaction between tannin and protein involves hydrogen bond formation between the protein amide carbonyl and the phenolic hydroxyl. (Hagerman A. E., Butler L. G. (1980), xe2x80x9cCondensed tannin purification and characterization of tannin-associated proteinxe2x80x9dJ. Agri Food Chem 28: 947-952.)
The interaction of tannic acid with protein is also pH-dependent, occurring preferably at pH values lower than the pKa of the phenolic groups, and related to its isoelectric point. The aromatic portion of the tannin may interact hydrophobically with non-polar amino acid side chains, such as phenylalanine, and these hydrophobic interactions are generally pH-dependent. The effects of the solvent composition on tannin-protein interactions suggest that complex formation results from hydrogen bonding and hydrophobic interactions. Studies on the interaction between condensed tannin and bovine serum albumin (BSA) showed that the complex includes strong non-covalent bonds. This complex can not be dissociated by strong buffers, but it can be disrupted by detergents or hydrogen bonding solvents.
Tannins have been used for the clarification of starch-containing solutions. The interaction of starch, an underivatized polysaccharide, with tannins was reported by Davis and Harvers (David,A. B. and Harbers, L. H.1974. xe2x80x9cHydrolysis of sorghum grains starch by rumen microorganisms and purified alpha-amylase was observed by electron Microscopyxe2x80x9d. J.Animal.Sci., 38:900). They reported that starch prepared by wet milling of bird resistant sorghum was less susceptible to the attack by enzymes than other starches. They suggested that absorption and retention of condensed tannins on starch might be responsible for this phenomenon. Tannins are known to associate with Sephadex(trademark) chromatographic gels. The complexation may be due to inclusion of phenolics within the pores of Sephade(trademark), interactions between oxygen atoms from ether groups that crosslink the gels, phenolic hydroxy groups as well as interactions between the phenyl ring acting as an electron donor and the hydroxy groups of gels (Brook,A. J. W. and Munday,K. C. 1970; xe2x80x9cInteractions of phenols, anilines and benzoic acids with Sephade(trademark) gelsxe2x80x9d. J. Chromatogr., 47:19.) Tannins have also been reported to have a strong affinity towards cyclodextrins, and polygalacturonate. Ozawa reported that starch, such as amylose, can develop a secondary structure containing hydrophobic cavities. Also polyamides, such as polyvinylpyrrolidone, non-ionic detergents, polyethylene oxides, and alpha, and beta cyclodextrins and alkaloids such as caffeine and cinchonine, associate strongly with polyphenol substrates (Ozawa, T. et.al. 1987. xe2x80x9cPolyphenol Interactions : Astringency and the loss of Astringency in ripening fruitxe2x80x9d. Phytochemistry, Vol. 26, N.11, pp.2937-2942.).
Non of the prior art to date has reported on the specific interactions of tannins with Aloe polysaccharides and protein bound-beta-glucans. Accordingly, the prior art has not overcome the disadvantageous deacetylation that generally occurs during processing of aloe polysaccharides as an acetylated poly-mannose polymer, free of bound malic acid and insoluble material and for the other cases of biologically active polysaccharides. The prior art neither discloses nor suggests that tannins can be used to precipitate the polysaccharides of aloe and especially the aloe acetylated mannose polysaccharides having the particular properties described herein. Further, the prior art does not disclose or suggest an aloe-derived polysaccharide having the properties described herein.
The present inventor has discovered that mucilaginous polysaccharides, such as the acetylated poly-mannose in aloe, and other similar polysaccharides such as beta-glucans produced by plants or poly-glucans produced by cultured microorganisms can be separated from solutions or aqueous extracts by complexation with tannins and specifically with hydrolyzable tannins. The present invention provides a superior method for the isolation of mucilaginous polysaccharides from a wide variety of plant and cell culture sources, especially those derived from aloe plants. The mucilaginous polysaccharides made according to the invention have properties that are improved over those mucilaginous polysaccharides made according to other known processes. The present invention, in particular, provides a process for isolating mucilaginous polysaccharides retaining most, if not all, of their native properties. The claimed invention also provides a process for the preparation of high quality mucilaginous polysaccharides in high yields. Further, the present process does not require large volumes of organic solvents as for the classical process employing ethanol, and in some embodiments, organic solvents are entirely eliminated from the process.
In one aspect the present invention is a method of isolating a mucilaginous polysaccharide comprising the steps of:
a) adding a first aqueous solution containing 0.5 to 10% weight/volume of hydrolizable or condensed tannins to a second aqueous solution containing polysaccharides or protein-bound polysaccharides while mixing to form a first insoluble complex composed of tannins and polysaccharides or biopolymers;
b) separating the complex from aqueous solutions to form a first water insoluble tannin-polysaccharide complex; and
c) breaking the tannin-biopolymer complex either by the use of solvents such as methanol, ethanol, methanol, butanol, acetone, 1,3 dioxalane or mixtures of these solvents with water with or without PEG and Tween 80; or
d) breaking the tannin-biopolymer complex by the use of water solutions containing either PVP, PPVP, casein, albumin, gelatin or other similar protein sources.
In some preferred embodiments, the solution of tannins is prepared by:
1) using an acetone: water extract made by gel permeation chromatography using Sephade(trademark) L-20 where commercially available tannic acid or other forms of Hydrolizable tannins are used as starting material. The ratio of acetone: water for elution of ellagotannins varies from 10: 90 up to 80: 20 and works best between 50:50 and 75:25; or
2) using an acetone : water extract made by classical chromatography using typical silica adsorbent materials and using tannic acid or other forms of tannins as starting material.
The present method can also comprise one or more of the following steps:
e) removing all tannins from the insoluble tannin-polysaccharide mass by the use of solvents to form a second mass of aloe biopolymers free of bounded tannins;
f) reducing the ionic strength of the Aloe gel extract by the use of specific resins prior the addition of the tannic acid;
g) dissolving the isolated tannin free polysaccharides in water to form a viscous solution containing the polysaccharide in a concentration ranging from 0.2% up to 0.6% weight/volume;
h) adding a preservative such as sodium benzoate or potassium sorbate to said aqueous solution;
i) reducing the particle size of solids in the first and/or second mass by means of special desintegrator/homogenizer equipment;
j) adding saline water, Tween 80, sodium sulfate, gallic acid or n-propyl-gallate to the second solution prior to the addition of the first solution containing tannins;
k) treating the first mass one or more times, optionally with mixing, with a sufficient amount of one or more solutions containing at least one of a surfactant and a glycol polymer to remove a major portion of the tannin from the mass to form a second mass having properties similar to the initial native polysaccharide;
1) using an acetone-water extract of ellagitannis made by treating commercially available tannic acid or other sources of tannins, with gel permeation chromatorgraphy or using classical chromatography techniques using other adsorbent type of packing materials, to complex the mucilaginous polysaccharides; and/or
m) using ellagitannins that have been extracted from the original tannic acid powder or other sources of tannic acid by the use or chromatography or gel permeation chromatography by using Sephadex(trademark) L-20. The ellagitannins extract contains at least one selected from the group consisting of the group chemically known as Nobotanins, Corilagins, Gemins, Augosin, Rugosin, Isorugosin, Corousilins, Coriariums, Ocnotheins, Agrimonin, Geraniin, Granatin and Cornusiins.
Another aspect of the invention provides a polysaccharide prepared according to the process described herein. In this aspect, the invention provides a polysaccharide isolated from plant extracts or produced by cell cultures, wherein the polysaccharide possesses properties which are about the same as those of the polysaccharide as it is found in the native plant or cell culture. In a preferred embodiment, the invention provides a polysaccharide isolated from an aloe plant, wherein the polysaccharide has at least one of a weight average molecular, an acetyl group: saccharide ratio, a saccharide content, a saccharide end group content, and a linkage group analysis similar to that of the polysaccharide as it is found in the native aloe plant.
The aloe-derived polysaccharide isolated according to the present invention generally possesses an acetyl group : saccharide ratio of about 1:1 or higher and is a high molecular weight polysaccharide. The molecular weight distribution of the isolated polysaccharide is generally broad and in the range of 10,000 up to 1,700,000. The aloe-derived polysaccharide isolated according to the present invention generally comprises arabinose, rhamnose, xylose, mannose, and glucose present in amounts similar to those found in the native plant, and preferably in amounts in the range of about 0.8-1.2% wt. of arabinose, 0.08-0.35% wt. of rhamnose, 0.35-045% wt. of xylose, 80-85% wt. of mannose, and 14-18% wt. of glucose based upon dry weight of the polysaccharide. The saccharide end group content and linkage group analysis of the polysaccharide isolated from aloe using the process of the invention generally yield the following results:
1.-Terminal arabinose (furanose): 0.7;
2.-Terminal xylose : 0.0;
3.-Terminal mannose: 0.9;
4.-Terminal galactose: 0.5;
5.-4-xylose: 0.7;
6.-4-mannose: 69.6;
7.-4-glucose: 9.7;
8.-3,4-mannose: 4.0;
9.-2,4-mannose: 2,5;
10.-2,3,6-mannose: 2,3;
11.-4,6-mannose: 5,4;
12.-4,6-glucose : 0.5;
13.-3,6-galactose: 1,4;
14.-3,4,6-mannose: 0.8;
15.-2,4,6-mannose: 0.8; and
16.-2,3,4,6-mannose: 0.7.
A variety of tannin compounds can be used in the process of the invention. Hydrolyzable tannins are preferred for the isolation of mucilaginous polysaccharides from aloe. Selection of a preferred tannin compound for isolating a particular type of polysaccharide generally depends upon the identity of the polysaccharide and the reaction conditions employed. Tannic acid is generally preferred for complexing mucilaginous polysaccharides from aloe.
Based upon the chemical affinity and specificity of the binding of condensed and hydrolyzable tannins to proteins and nitrogen acetyl functional groups present in mucilaginous polysaccharides, a particular polyphenol, proanthocyanin, gallatannins, ellagitannins (referred to as hydrolyzable tannins) will be preferred for isolation of the mucilaginous polysaccharides. In general about 1 gram of Aloe polysaccharide in solution will bind about 0.5 to 1 gram of tannic acid. However, this relation is not the same when an acetone : water 50:50 extract of Ellagotannins eluted out of a Sephadex(trademark) LH-20 column is used instead. In the latter case, about 1 gram of the Aloe biopolymer network will generally bind with about 0.02 to 0.3 g. of ellagitannins. The ratio in which tannins bind to specific polysaccharide depends upon many varibles and a stoichiometric relationis not required.
The polysaccharides isolated from plants, cereals, fungi or cell cultures according to the invention are used in a wide range of products. Accordingly, the present invention provides a composition comprising an aloe-derived polysaccharide isolated by a process using a tannin, said composition being present in at least one of a beverage, candy, comestible, tonic, lotion, cosmetic, pharmaceutical composition, suppository, implant, shampoo, hair conditioner, wound dressing, wound or injury treatment product, anti-itch formulation, sun-burn formulation, topical formulation, oral formulation, dietary composition, food supplement, injectable formulation and other products known to those of skill in the aloe art.
Other features, advantages and embodiments of the invention will be apparent to those of ordinary skill in the art from the following description, examples and appended claims.